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Mouse and Rat Anesthesia and Analgesia

Judith A. Davis¹

¹NIAAA/NIH, Rockville, Maryland

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Appendix 4B

DOI:

10.1002/0471142301.nsa04bs42

Online Posting Date:

January, 2008

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Abstract

Many animal models used in neuroscience research must be surgically created and/or anesthetized for imaging studies. The purpose of this unit is to review the advantages and disadvantages of various anesthetic and analgesic agents in rodents; to discuss state-of-the-art methods for monitoring anesthesia; and to provide tips for troubleshooting problems with anesthesia. Curr. Protoc. Neurosci. 42:A.4B.1-A.4B.21. © 2008 by John Wiley & Sons, Inc.

Keywords: mouse; rat; anesthesia; analgesia

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Introduction
Basic Protocol 1: Injectable Anesthesia for Mouse and Rat
Basic Protocol 2: Inhalant Anesthesia Using Isoflurane for Mouse and Rat
Basic Protocol 3: Analgesia for Mice and Rats
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Injectable Anesthesia for Mouse and Rat

Materials

Laboratory mice or rats
Anesthetic of choice (see Table A.4B.2)
Petroleum-based artificial tear ointment
Lactated Ringer's solution, warmed to 35° to 36°C (optional; e.g., A.J. Buck or J.A. Webster)

For the surgeon:
- Clean laboratory coat
- Face mask
- Head cover (optional)
- Sterile gloves
Warm water recirculating heating pad or heat lamp, 38° ± 2°C
Sterile drape (optional)
Cages for mice or rats
Laboratory scale or balance (capacity 800 g; accuracy 0.1 g)
1- or 3-ml syringe
20- or 22-G needle
21- or 23-G needle (optional)

Additional reagents and equipment for intraperitoneal and subcutaneous injection (appendix 4F)

Table A.4B.2 Guidelines for Injectable Anesthetics in Rodents

Agent	Species	Dosage (mg/kg) and route of administration^a	Duration of surgical anesthesia (min)^b

Pentobarbital^c	Mouse	40-70, i.p.	20-40
	Rat	30-50, i.p.	15-60
Ketamine/xylazine^d	Mouse	60-100 ketamine + 5-7.5 xylazine, i.p.	20-25
	Rat	50-90 ketamine + 5-10 xylazine, i.p.	60-80
Ketamine/xylazine/acepromazine^e	Mouse	25-30, i.p.	20-35
	Rat	30-40, i.p.	20-45
Ketamine/medetomidine^f	Mouse	NA	NA
	Rat	75 ketamine + 0.5 medetomidine, i.p.	15-60
Tribromoethanol^g	Mouse	125-160, i.p.	15-30
	Rat	300, i.p.	15-20
Atropine^h	Mouse	0.02-0.05 mg/kg, s.c., i.p.	30-40
	Rat	0.04 mg/kg, s.c., i.p.	15-20
Bupivicaine (Marcaine)ⁱ	Mouse	0.5% solution. Local infiltration or drop-wise administration to incision site.	NA
	Rat		NA

^aDosages are from a variety of sources. The dose may need to be adjusted for individual situations. Abbreviations: i.p., intraperitoneally; s.c., subcutaneously.

^bDuration of surgical anesthesia (unconscious, nonresponsive to painful stimuli) is not the same as “sleep time” (quiet, not moving, but responsive to stimuli), which is generally much longer.

^cDilute 1:10 (v/v) with sterile saline. This is a controlled substance. Barbiturate classification (long, short, ultrashort) is misleading, as species differences in barbiturate pharmacokinetics are responsible for significant variation in duration of action. As barbiturates do not provide analgesia, they are often combined with sedatives or tranquilizers to provide deep anesthesia and smooth recovery.

^dKetamine and xylazine can be safely mixed and given as a single injection. After mixing, dilute 1:10 (v/v) with sterile saline. Ketamine is a controlled drug. Caution: Some strains of rats are at increased risk for developing post-anesthetic corneal lesions with this drug combination (Turner and Albassam, 2005).

^eMix 1.5 ml (100 mg/ml) ketamine, 1.5 ml (20 mg/ml) xylazine, and 0.5 ml (2 mg/ml) acepromazine together; stable at room temperature (shelf life of the ingredients). Do not use in preweanling animals. Ketamine is a controlled drug.

^fKetamine/medetomidine provides marked differences in surgical anesthesia between male and female mice and rats. Male mice require less ketamine whereas female mice require a higher dose of ketamine to effect loss of righting reflex. The loss of righting reflex is also gender specific, occurring more rapidly in males than in females. Males have loss of reflexes for 25-60 min; females, 140-150 min. Sleep time is also marked; males 135-160 min, females 240-300 min. Heavy urination occurs, which may lead to dehydration, indicating the need for parenteral fluids. Wetting of fur may also lead to hypothermia. Ketamine is a controlled drug. NA, data not available. The effects of this drug combination can be reversed with atipamezole (Antisedan, Pfizer). CAUTION: This drug combination does not produce an adequate plane of anesthesia for surgery! It does provide adequate chemical restraint (Cruz et al., 1998).

^gMake a 100% (w/v) stock solution by dissolving 5 g of 2,2,2-tribromoethanol in 5 ml 2-methyl-2-butanol (tert-amyl alcohol). Gentle heating (50°C) provides better solubility. The anesthetic solution should be freshly prepared from the stock solution by adding 1.25 ml of stock solution to 48.75 ml of sterile saline. Both stock and anesthetic solutions are stable 2 to 4 months at 4°C in a dark bottle. Anesthetic solutions should be made fresh weekly; filter the solution using a 0.2-µm filter. CAUTION: Stored solutions often deteriorate to irritant solutions that cause peritonitis and/or death following i.p. administration. Add 1 drop of Congo Red (0.1% w/v) to 5 ml of anesthetic solution. Purple color developing at pH < 5 indicates decomposition (Papaioannou and Fox, 1993).

^hAtropine is an anticholinergic, not an anesthetic. Atropine blocks acetylcholine at muscarinic receptors. Desirable effects include reduction in bronchial secretions and protection of the heart from vagal stimulation, which may occur during surgical procedures. If used, it should be given 5 to 10 min before the anesthetic agent.

ⁱBupivicaine applied to the incision site during or after closure of the incision augments analgesia by providing local anesthesia at the site of the incision. The local anesthetic effect may last for 10-12 hr.

Basic Protocol 2: Inhalant Anesthesia Using Isoflurane for Mouse and Rat

Materials

Isoflurane
Laboratory mice or rats
Petroleum-based artificial tear ointment
Lactated Ringer's solution, warmed to 35° to 36°C (optional)

For the surgeon:
- Clean laboratory coat
- Face mask
- Head cover (optional)
- Sterile gloves
6-, 20-, or 30-ml syringe (optional)
Latex glove (optional)
30- or 60-ml syringe case cover and cap
3/8–in. brass tube sleeve insert (optional; Anderson Barrows PB96760PT, no. 25080)
3/8-in. (i.d.) vinyl or polyethylene tubing (optional)
#6–32, 2 in. machine screw (optional)
Commercial rodent face mask (optional; Kent Scientific or SurgiVet)
Warm water recirculating heating pad or heat lamp, 35° ± 1°C
Sterile drape (optional)
Precision vaporizer (Kent Scientific or SurgiVet)
Oxygen tank with flowmeter (Kent Scientific or SurgiVet)
Induction chamber (20-cm length × 10-cm height × 10-cm width; 2 liters) with inlet and outlet ports
Additional reagents and equipment for subcutaneous injection (appendix 4F)

All procedures with isoflurane require a scavenging device for expired anesthetic gases. Frequently, a chemical fume hood, down draft table, or safety cabinet is used for continuous exhaust of anesthetic gases away from personnel (see Critical Parameters).

Looking for materials? Suppliers Guide

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Figures

Figure A.4B.1

Assembly of a simple face mask for use in rodent anesthesia. (A) Syringe with attached rubber glove finger. (B) Hole cut in glove finger tip to fit over animal's muzzle.

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Figure A.4B.2

Components, made from readily available equipment, needed to make a rat face mask to use with a stereotaxic apparatus. A similar mouse face mask can be made using smaller syringe case components.

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Figure A.4B.3

The face mask assembled and attached to the stereotaxic apparatus. The anesthetized animal's nose can now be placed through the diaphragm, positioning the animal's upper incisors through the incisor hole of the mouth bar.

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Figure A.4B.4

Animal with face mask.

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Literature Cited

Literature Cited
	Abbott, F.V. and Bonder, M. 1997. Options for management of acute pain in the rat. Vet. Rec. 140:553-557.
	Ahmed, F., Lundin, G.G., and Shire, J.G.M. 1989. Lysosomal mutations increase susceptibility to anesthetics. Experientia 45:1133-1135.
	Alexander, C.M., Teller, L.E., and Gross, J.B. 1989. Principles of pulse oximetry: Theoretical and practical considerations. Anesth. Analg. 68:368-376.
	Basbaum, A.I. and Levine, J.D. 1991. Opiate analgesia. N. Engl. J. Med. 325:1168-1169.
	Clark, J.A., Myers, P.H., Goelz, M.F., Thigpen, J.E., and Forsythe, D.B. 1997. Pica behavior associated with buprenorphine administration in the rat. Lab. Anim. Sci. 47:300-303.
	Cooper, D.M., DeLong, D., and Gillett, C.S. 1997. Analgesic efficacy of acetaminophen and buprenorphine administered in drinking water of rats. Contemp. Top. Lab. Anim. Sci. 36:58-62.
	Cruz, J.I., Loste, J.M., and Burzaco, O.H. 1998. Observations on the use of medetomidine/ketamine and its reversal with atipamezole for chemical restraint in the mouse. Lab. Anim. 32:18-22.
	Flecknell, P.A. 1996. Laboratory Animal Anesthesia, 2nd ed. Academic Press, London.
	Flecknell, P.A. 1998. Analgesia in small mammals. Semin. Avian Exotic Pet Med. 7:41-47.
	Flecknell, P.A., Kirk, A.J.B., Fox, C.E., and Dark, J.H. 1990. Long-term anesthesia with propofol and alfentanil in the dog and its partial reversal with nalbuphine. J. Assoc. Vet. Anesth. 17:11-16.
	Flecknell, P.A., Roughan, J.V., and Stewart, R. 1999. Use of oral buprenorphine (“buprenorphine jello”) for postoperative analgesia in rats—a clinical trial. Lab. Anim. 33:169-174.
	Gwynne, B.J. and Wallace, J. 1992. A modified anesthetic induction chamber for rats. Lab. Anim. 26:163-166.
	Hecker, B.R., Lake, C.L., DiFazio, C.A., Moscicki, J.C., and Engle, J.S. 1983. The decrease in minimum alveolar concentration produced by sufentanil in rats. Anesth. Analg. 62:987-990.
	Kufoy, E.A., Vytautas, A.P., Parks, C.D., Wells, A., Yang, C., and Fox, A. 1989. Keratoconjunctivitis sicca with associated secondary uveitis elicited in rats after systemic xylazine/ketamine anesthesia. Exp. Eye Res. 49:861-871.
	Liles, J.H. and Flecknell, P.A. 1993. The effects of surgical stimulus on the rat and the influence of analgesic treatment. Br. Vet. J. 149:515-525.
	Liles, J.H., Flecknell, P.A., Roughan, J., and Cruz-Madorran, I. 1988. Influence of oral buprenorphine, oral naltrexone or morphine on the effects of laparotomy in the rat. Lab. Anim. 32:149-161.
	Lovell, D.P. 1986a. Variation in pentobarbitone sleep time in mice. 1. Strain and sex differences. Lab. Anim. 20:85-90.
	Lovell, D.P. 1986b. Variation in pentobarbitone sleep time in mice. 2. Variables affecting test results. Lab. Anim. 20:91-96.
	Lovell, D.P. 1986c. Variation in barbiturate sleeping time in mice. 3. Strain X environment interactions. Lab. Anim. 20:307-312.
	Papaioannou, V.E. and Fox, J.G. 1993. Efficacy of tribromoethanol anesthesia in mice. Lab. Anim. Sci. 43:189-192.
	Smith, W. 1993. Responses of laboratory animals to some injectable anesthetics. Lab. Anim. 27:30-39.
	Thurmon, J.C., Tranquilli, W.J., and Benson, G.J. 1996. Preanesthetics and anesthetic adjuncts. In Lumb and Jones' Veterinary Anesthesia, 3rd ed. (J.C. Thurmon, W.J. Tranquilli, and G.J. Benson, eds.) pp. 183-209. Williams & Wilkins, Baltimore.
	Turner, P.V. and Albassam, M.A. 2005. Susceptibility of rats to corneal lesions after injectable anesthesia. Comp. Med. 55:175-182.
	Vender, J.R., Hand, C.M., Sedor, D., Tabor, S.L., and Black, P. 1995. Oxygen saturation monitoring in experimental surgery: A comparison of pulse oximetry and arterial blood gas measurement. Lab. Anim. Sci. 45:211-215.
Key References
	Bishop, Y.(ed). 1998. The Veterinary Formulary, 4th ed. Pharmaceutical Press, London.
Provides dosages for a wide range of drugs, including antibiotics, for commonly used laboratory animals.
	Dorsch, J.A. and Dorsch, S.E. 1999. Understanding Anesthesia Equipment, 4th ed. Williams & Wilkins, Baltimore.
This text is the most comprehensive source of information for equipment used in the United States.
	Flecknell, 1996. See above.
This book offers practical advice for anesthesia and analgesia of laboratory animals. It is considered a “must have” for laboratory animal veterinarians.
	Kohn, D.F., Wixson, S.K., White, W.J., and Benson, G.J. (eds). 1997. Anesthesia and Analgesia in Laboratory Animals. American College of Laboratory Animal Medicine Series, Academic Press, New York.
This is an overall excellent reference.
Internet Resources
	http://www.kentscientific.com
This is an excellent site for finding information about equipment mentioned in this unit, as well as finding other resources. Examples of major headings: Neuroscience, Cardiovascular, Respiratory Parameters, Data Acquisition, Recording Devices, Perfusion. The company's personnel are excellent at working with investigators to develop customized equipment.
	http://www.surgivet.com
Another excellent site for all product information related to surgery. For example, several different types of scavenging systems for waste gas evacuation can be found at this site.
	http://www.med-e-cell.com
An excellent site for different sources of items such as catheters and infusion pumps.
	http://www.innovrsch.com/index.html
This site is provided for investigators wishing to explore alternative methods to deliver long-term drugs, analgesics, and so on, in time-release form to avoid repeated injections and maintain therapeutic concentrations.
	http://www.Bio-Serv.com
An excellent resource for customized (and certified) diets (all species). They also specialize in enrichment treats and medicated dosing systems.